Salmonella typhi, the causative bacterium for typhoid fever in human beings is a major endemic disease in Africa, Asia, and Middle East. Food and water contaminated with S. typhi bacterium was identified as major source in transmission of the disease. Various studies have shown that the global burden of typhoid fever varies in different parts of the world. More than 100 cases in 100,000 populations per year reported in South Central Asia and South-East Asia; Asia, Africa, Latin America and the Caribbean are estimated to have medium incidence of typhoid fever, i.e., 10 to 100 cases in 100,000 populations per year; New Zealand, Australia and Europe have low to very low incidence (Crump et al., 2004). This suggests that Typhoid fever is strongly endemic in the regions of the World particularly in the developing nations and countries with low resource settings.
Salmonella belongs to the family of Enterobacteriaceae that includes the genera Shigella, Escherichia, and Vibrio. The genus of Salmonella contains two different species, S. enterica and S. bongori. S. enterica is further divided into six subspecies (enterica, salamae, arizonae, diarizonae, houtenae and indica) containing 2443 serovars. The agents that cause enteric fever are therefore Salmonella enterica subspecies enterica serovar typhi (commonly referred to as S. enterica serovar typhi) and serovars Paratyphi A, B and C. A serovar or serotype can be defined as a strain that has a unique surface molecule which is responsible for the production of specific antibody. Each serotype has subtle chemical differences in their antigenic region (Brenner et al., 2000).
Salmonella typhi has a combination of characteristics that make it an effective pathogen. This species contains an endotoxin typical of gram negative organisms, as well as the Vi polysaccharide antigen which is thought to increase virulence. It also produces and excretes a protein known as “invasin” that allows non-phagocytic cells to take up the bacterium, allowing it to live intracellularly. It is also able to inhibit the oxidative burst of leukocytes, making innate immune response ineffective. During the last decade, Salmonella species have been found to acquire more and more antibiotic resistance. The cause appears to be the increased and indiscriminate use of antibiotics in the treatment of Salmonellosis of humans and animals, and the addition of growth-promoting antibiotics to the food of breeding animals. Plasmid-borne antibiotic resistance is very frequent among Salmonella strains involved in pediatric epidemics. Resistance to ampicillin, streptomycin, kanamycin, chloramphenicol, tetracycline, ceftriaxone, cefotaxine, cefoperazone and sulfonamides is commonly observed Colistin-resistance has not yet been observed. Salmonella strains should be systematically checked for antibiotic resistance to aid in the choice of an efficient drug when needed and to detect any change in antibiotic susceptibility of strains (either from animal or human source). Until 1972, Salmonella typhi strains had remained susceptible to antibiotics, including chloramphenicol (the antibiotic most commonly used against typhoid); but in 1972 a widespread epidemic in Mexico was caused by a chloramphenicol resistant strain of Salmonella typhi. Other chloramphenicol-resistant strains have since been isolated in India, Thailand and Vietnam.
Vaccination against typhoid fever caused due to Salmonella Typhi is essential for protection against these life-threatening disease due to increasing antibiotic resistance. It is also an important protective tool for people travelling into areas where typhoid fever is endemic. As the bacterium has the ability to acquire multi-drug resistance ability, antibiotics may not offer complete protection. Three types of typhoid vaccines have been made currently available for use till now: (1) Parenteral killed whole cell vaccine; (2) Oral live-attenuated vaccine; and (3) Typhoid-Vi capsular polysaccharide vaccine for parenteral use. Vaccines against typhoid fever were designed in early ages when the organism's cellular and molecular complexity was studied clearly. Initially parenterally administered whole cell S. typhi killed by heat-phenol-inactivation method was used as a vaccine, to be administered in two doses. Since the whole cell inactivated vaccines contain the ‘O’ antigen (endotoxin), they tend to produce local and general reactions in vaccinated individuals and these types of vaccines required a booster dose for every two years. Oral live-attenuated Ty21a vaccine are considered as second generation vaccines prepared with mutant S. typhi strain lacking adenylate-cyclase and AMP receptor protein and mutants auxotrophic for p-amino benzoate and adenine. These live attenuated vaccines reported poor efficacy and was found to be not suitable for administration of children's below 6 years of age. Additionally, a booster dose is also required for every 5 years. Subsequently, capsular Vi-polysaccharide of S. typhi was identified as a protective immunogen capable for eliciting adequate immune responses in humans and hence used as a potential vaccine candidate in routine immunization schedule. A dose of 25 μg/0.5 mL injection of purified capsular Vi-polysaccharide (ViPs) can produce maximum sero-conversion i.e. fourfold rise in antibodies. However, the limitations of the Vi-polysaccharide vaccine have been reported in many clinical trials that native polysaccharide vaccine are incapable or do not produce secondary memory responses. This phenomenon is because of bacterial polysaccharides are T-cell independent in nature and hence are not capable to produce cell mediated immune responses. Therefore to overcome the said problem, polysaccharides of S. typhi and carrier proteins were further conjugated to form polysaccharide-protein molecules to make it T-cell dependent antigens. There are various factors that influence the coupling of polysaccharides and proteins which depend upon molecular weight of the ViPs and carrier proteins selected and activation of the functional groups. Low molecular weight polysaccharides can result in efficient coupling to carrier proteins. Different carrier proteins like tetanus toxoid, diphtheria CRM 197, the B subunit of the heat-labile toxin (LT-B) of Escherichia coli, the recombinant exoprotein A (rEPA) of Pseudomonas aeruginosa, Flagellin Fli C, and Horseshoe rab Haemocyanin (HCH) have been mostly used for conjugation.
WO1996/011709 discloses an O-acetylated oligonucleotide or polygalactouronate pectin which is substantially identical to Vi polysaccharide subunit structure conjugated to a carrier protein tetanus toxoid wherein the carrier protein being derivatized with cystamine. This particular patent teaches to conjugate an identical polysaccharide but not Vi-polysaccharide to carrier protein with a different derivatizer that is cystamine, Subsequently, WO1998/026799 discloses an isolated lipo-polysaccharide from Salmonella Paratyphi A, having removed its Lipid A through detoxification and retaining its O-acetyl content between 70% to 80% and then conjugated to a carrier protein tetanus toxoid through adipic acid dihydrazide (ADH). WO2000/033882 discloses a Vi-polysaccharide of the Salmonella typhi covalently bound to a protein Pseudomonas aeruginosa (Vi-rEPA) conjugate through adipic acid dihydrazide. WO2007/039917 discloses an exogenous antigen of Salmonella typhi which is covalently/non-covalently bonded to a Heat Shock Protein.
WO2009/150543 describes a conjugated Vi-polysaccharide to be used as a vaccine composition against Salmonella typhi causing typhoid fever, wherein the Vi-polysaccharide is covalently conjugated to a protein selected from CRM197 or tetanus toxoid. The method of conjugation as disclosed in WO2009/150543 includes first simultaneously adding carrier protein which is preferably CRM197 or tetanus toxoid to a linker such as adipic acid dihydrazide (ADH), and a carbodiimide such as 1-ethyl-3(3-dimethylaminopropyl) carbodiimide (EDAC), to give a derivatized carrier protein in presence of a 2-(N-morpholino) ethane sulphonic acid (MES buffer). The weight ratio of the carbodiimide EDAC to the carrier protein is between 0.1 to 0.15. It also discloses that higher amounts of carbodiimide/protein ratios can cause aggregate formation. Derivatization of the carrier protein is followed by activation of the Vi-polysaccharide (ViPs) as well. The Vi-polysaccharide is also activated with a carbodiimide wherein various ratios of ViPs and carbodiimide (EDAC) are mixed to activate the Vi-polysaccharide. It is mentioned that Vi activation can be performed at room temperature within 2 minutes wherein higher ratios between 1.5:1 to 200:1 can be used. The derivatized carrier protein CRM197 or tetanus toxoid and the activated Vi polysaccharide of Salmonella typhi is then reacted with each other to get the conjugated ViPs-CRM197 or ViPs-TT conjugate, followed by removal of the excess linker.
Safety and immunogenicity of ViPs conjugate vaccines in adults, teenagers and 2- to 4-year old children in Vietnam were evaluated by Zuzana Kossaczka et al in 1999. In this study the geometric mean level of anti-Vi-rEPA (conjugate vaccine) in the 2- to 4-year old children was higher than that elicited by Vi capsular polysaccharide vaccine in the 5 to 14 years old children. Re-injection of conjugate vaccine induced rise in antibody titers in 2- to 4-year old children (T-cell dependent). Konadu et al. (2000) prepared S. paratyphi A O-specific polysaccharide (O-SP) and coupled to tetanus toxoid. These conjugates elicited IgG antibodies in mice and the safety and immunogenicity of the conjugates was evaluated in Vietnamese adults, teenagers and 2-4 years old children. The study concluded that these experimental conjugates were safer and proven to elicit IgG antibodies in adults, teenagers and 2-4 years old children. The efficacy of Salmonella typhi ViPs conjugate vaccine in two to five-year old children was evaluated by Feng Ying C et al. In this study the conjugate typhoid vaccine was found to be safe and immunogenic and had more than 90% efficacy in children two to five years old. Serum IgG Vi antibodies after six weeks of second dose levels increased 10 fold in 36 evaluated children. These cases were followed for a period of 27 months. No serious adverse reactions were observed in the study due to the vaccination. Effect of dosage on immunogenicity of ViPs conjugate vaccine injected twice in to 2 to 5 years old Vietnamese children was studied by Do Gia Canh et al. In this study dosage immunogenicity study of 5 μg, 12.5 μg and 25 μg of conjugate vaccine injected twice, six weeks apart was evaluated. This study also confirmed the safety and consistent immunogenicity of four lots of conjugate vaccine in this and previous trials. Novartis vaccine institute for global health carried-out three different dose-related formulations of ViPs-CRM197. They carried out different doses were 25 μg, 12.5 μg, 5 μg and 1.25 μg/dose. The GMT for these concentration at day 28 was 304 U (units), 192 U, 111 U and 63 U respectively. At day 28 GMT with 25 μg/dose elicited the highest antibody level (304 U) after single injection.
Although, the present state of the art includes conjugate vaccines with Vi-polysaccharide and a carrier protein, however, the existing native Vi-polysaccharide conjugate vaccines when tested in many human clinical trials revealed that these vaccines are safe and immunogenic in adults but failed to induce any protective immune response in children below 2 years of age. Therefore, this native S. typhi polysaccharide vaccine did not prove to find any particular solution against deadly S. typhi infections in children's less than 2 years of age which demands a new vaccine which could immunize children of age below 2 years against S. typhi infections responsible for causing typhoid. The age group of below 2 years of age is the most prone to infections by Salmonella typhi but there seems to be presently not available to the mankind any protective vaccine against S. typhii for infants below 2 years of age still now. As discussed above, various carrier proteins such as CRM-197, r-EPA, have been conjugated to Vi-polysaccharide, wherein the Vi polysaccharide might not have been isolated from S. typhi, or being depicted from any other sources. Producing typhoid conjugate vaccines is therefore, specific to the particular carrier protein involved and the native polysaccharide involved in the conjugation process and the resulting conjugate vaccine. Each carrier protein-polysaccharide conjugation makes itself a different identity of conjugate vaccine. The prior arts disclosed in the area of typhoid conjugate vaccine, methodology and as well as those currently used, have their own drawbacks, which might be a possible reason behind not having any conjugate vaccine presently available which can protect children below 2 years of age.
It is also very much evident and well known in the current state of the art that, the present typhoid conjugate vaccines require at least 2 or more injections with a time interval of 6-8 weeks to comprise a complete vaccination schedule A typhoid Vi capsular polysaccharide-tetanus toxoid (ViPs-TT) conjugate vaccine was made available to the public by BioMed, which required 2 injections of 5 μg each with a time interval of 6-8 weeks to complete a single vaccination schedule. However, this ViPs-TT vaccine also was not capable to immunize children below 2 years of age against Salmonella Typhii. 
Hence, there exists a need of alternating conjugation methodologies, which would reduce costs, and the number of injections to only one injection capable of eliciting sufficient immune response and other associated technical concerns in the field of conjugation chemistry which would be simpler, less time consuming, cost-effective and safe. An efficient vaccine must be capable of triggering a good immune response and must be applicable for use in infants especially below 2 years of age. The disclosure as set forth in this invention attributes to novel alternative methods of conjugating the Vi polysaccharide along with the specific carrier protein tetanus toxoid (TT) in an inventive manner put-forth in this application which potentially overcomes the drawbacks of native polysaccharide vaccines and also current conjugation methodologies including other ViPs vaccines conjugated to carrier proteins. The Vi-polysaccharide-protein conjugate vaccine produced by this particular methodology as set forth in this patent application makes it more suitable for immunization in children and infants including less than 2 years of age with secondary memory responses producing high affinity antibodies against S. typhi infections, including humans of any age group. It is also another advantage of the invention put forth in this application that, the number of injections of typhoid conjugate vaccine to complete a vaccination schedule has also been reduced to only ONCE, which at the same time elicits a better immune response when compared to immune response generated by a vaccination schedule of 2 or 3 injections of typhoid conjugate vaccine being practiced earlier. Single injection of typhoid conjugate vaccine is always preferable for infants and children since it would reduce, additional visits to the clinic, pain suffered by a child or infant for repeated injections for vaccination. It is already reported that, 40% of injections worldwide are administered with un-sterilized, reused syringes and needles, and especially in the targeted developing countries, this proportion is more than 70%, exposing millions of people to infections wherein pathogens enter the tissues of the body during an injection. Furthermore, poor collection and disposal of dirty injection equipment, exposes healthcare workers and the community to the risk of needle stick injuries. Unfortunately, in some countries, unsafe disposal also leads to re-sale of used equipment on the black market. Open burning of syringes is unsafe under WHO, yet half of the non-industrialized countries in the World, follow this practice. (“Injection safety”, Health Topics A to Z. World Health Organization. Retrieved May 9, 2011). Unsafe injections cause an estimated 1.3 million early deaths each year. (M. A. Miller & E. Pisani. “The cost of unsafe injections”. Bulletin of the World Health Organization 77 (10): 1808-811). Although, to improve injection safety, the WHO recommends certain alternatives to injections subject to availability, or else controlling and regulating the activity of health care workers and patients, vaccinees, by ensuring the availability of equipment and supplies aided with managing waste safely and appropriately; these measures are not always possible to be observed absolutely. In such circumstances, a combination of Typhoid conjugate and measles vaccine in one SINGLE shot will definitely play a substantial role in decrease of worries pertaining to injection safety in national immunization programs. Many countries do have legislation or policies that mandate that healthcare professionals use a safety syringe (safety engineered needle) or alternative methods of administering medicines whenever possible, however reduction in the number of injections for ensuring protection against Typhoid and Measles in one single injection in infants surely indicates high compliance from a public health perspective since where there was at least 3 injections required earlier to inject typhoid (2 injections minimum) and measles (one injection) vaccine, now the same is accomplished by only ONE injection.